KR100502523B1 - Analysis of predisposition based on human airway tripsin proteaxe gene polymorphism - Google Patents

Abstract

A method for predicting the constitution susceptible to the onset of specific diseases in individual humans, for example, respiratory diseases such as chronic obstructive pulmonary diseases, sinobronchial syndrome, pulmonary emphysema, diffuse panbronchiolitis or bronchiectasis, effects of treatment on patients or prognosis of the treatment by analyzing the genetic polymorphisms of a human trypsin-like enzyme of the respiratory tract.

Description

ANALYSIS OF PREDISPOSITION BASED ON HUMAN AIRWAY TRIPSIN PROTEAXE GENE POLYMORPHISM}

The present invention relates to a method for predicting the effect on the constitution and the method of treating a disease patient, or a method for predicting the prognosis of the disease, by which a polymorphism of a human airway trypsin form enzyme is prone to develop. It is about.

In recent years, studies have been conducted on genes that are related not only to genetic diseases caused by single gene deletion and mutation but also to multifactorial diseases in which several gene traits and environmental factors are intertwined. As a result, deficiencies and point mutations in the genes involved in these multifactorial diseases, as well as variations in the isoforms and even the gene parts (introns, promoters) that do not affect the actual translated amino acid sequences, are considered risk factors for the disease. have.

In the prior art, it has been reported that the correlation between bone density and the intron gene polymorphism of the vitamin D receptor is recognized in the field of bone disease (Morrison, N. A. et al., Nature, 367: 284-287, 1994). In the circulatory field, polymorphisms of type I (insertion type) and type D (defective) of angiotensin converting enzymes develop myocardial infarction (Cambiem, F. et al., Nature, 359: 641-644, 1992). And an amino acid substitution of M235T of angiotensinogen and a polymorphism in the promoter region of G-6A are associated with intrinsic hypertension (Inoue, I. et al., J. Clin. Invest., 99 1786-1797, 1997). In the field of the nervous system, the association between the onset of dementia and the isoform of apoE protein has been reported. In cancer-related studies, many studies have been made on the relationship between the genetic polymorphism of glutathione S transferase and the onset of cancer. In the field of respiratory diseases, the onset and condition of asthma, TNF (Moffatt, MF et al., Hum. Mol. Genet. 6 (4): 551-554, 1997) and angiotensin converting enzymes (Benessiano, J. et al., J. Allergy Clin. Immunol. 99 (1): 53-57, 1997) has been reported to relate to the polymorphism of the gene.

In addition, the genetic background of attention to the sensitivity of the drug used to treat the disease as one of the different causes by the patient, the direction that the treatment method according to the sensitivity of the individual drug is selected from the medical field by the gene polymorphism diagnosis. have. In addition, it is considered that it is effective to select a patient group for which a drug is expected to be effective in clinical trials and to develop a drug (Kleyn K. W. et al .: Science, 281: 1820-1821, 1998). Conventional reports on drug susceptibility and gene polymorphisms include the effects of angiotensin converting enzyme (ACE) on the intron gene polymorphism and the effect of ACE inhibitors (Yoshida, H. et al., J. Clin.Invest). 96: 2162-2169, 1995) and on the effect of beta-agonists on beta 2-adrenergic receptor gene polymorphism and on asthma (Liggett, SB, Am. J. Respir. Crit. Care Med. 156 (4 Pt 2): S156-162, 1997).

On the other hand, the human airway trypsin form enzyme which concerns on this invention is refine | purified in the sputum of chronic airway disease patients (Japanese Unexamined-Japanese-Patent No. 7-067640 and Yasuoka S. et al., Am. J. Respir. Cell Mol. Biol., 16: 300-308, 1997), its amino acid sequences and cDNA sequences have already been clarified (Japanese Patent Laid-Open No. 8-89246 and Yamaoka K. et al., J. Biol. Chem. , 273 (19): 11895-11901, 1998). The activity of this enzyme has been studied to some extent in vitro. Conditions of Airway Inflammation from Involvement in Mucociliary Movement and Increasing Cytokines Production of IL-8, GM-CSF, etc. from Human Bronchial Epithelial Cell Lines (Terrao Noriko et al., Japanese Respiratory Society of 1998) The possibility of involvement in the furnace is considered. In addition, enzyme activity such as fibrinogen degrading activity and plasminogen activating activity (pro-urokinase) activation (Conference on Proteases and Inhibitors in Pathophysiology and Therapeutics in Yoshinaga et al., 1998 It is anticipated that it may act anti-inflammatory through fibrin formation on airway mucosal surfaces, or that the condition may be modified in chronic airway diseases, and that it may be involved in cancer metastasis. However, the involvement of this enzyme in physiological functions and conditions in vivo has not yet been fully elucidated, and there is no known gene part (intron, promoter) that does not correspond to the amino acid sequence to be actually translated. . In addition, the presence of the gene polymorphism and the association between the gene polymorphism and the disease for this human airway trypsin form enzyme has not been investigated until now.

However, predicting disease-related constitution by genetic analysis can give a lot of information to predict the onset of a specific disease, to predict the prognosis of the treatment, to select an appropriate treatment method and a dosage agent. Therefore, it is desired by many doctors and patients, and since these enable prevention and early treatment of the onset, it is thought that it will be necessary for future medical care, leading to a reduction of a large amount of medical expenses.

However, it is very difficult to find a gene related to a disease and establish a method of interpretation thereof. As described above, there are few examples of gene analysis methods in which association with a disease is recognized. Therefore, there is a strong demand for the development of genetic analysis methods that can predict various disease-related constitutions.

On the other hand, which diseases are related to human airway trypsin form enzymes are not yet elucidated.

FIG. 1 shows agarose gel electrophoresis pattern of DNA fragments comprising intron regions of genes encoding mature proteins of human airway trypsin form enzymes obtained by gene amplification. Lanes 1 and 5 are markers (10 Kb, 7 Kb, 5 Kb, 4 Kb, 3 Kb, 2.5 Kb, 2 Kb, 1.5 Kb, 1 Kb) from the top, lanes 2 are primers A1 (SEQ ID NO: 7) and A2 ( DNA fragment of about 6 Kb amplified by SEQ ID NO: 8), lane 3 is about 1.5 Kb DNA fragment amplified by primers B1 (SEQ ID NO: 9) and B2 (SEQ ID NO: 10), and lane 4 is primer About 3.4 Kb of DNA fragments amplified by C1 (SEQ ID NO: 11) and C2 (SEQ ID NO: 12) were observed.

Fig. 2 shows agarose gel electrophoresis pattern of DNA fragments containing intron regions of genes encoding the propeptide of human airway trypsin form enzymes obtained by gene amplification. Lanes 1 and 4 are markers (10 Kb, 7 Kb, 5 Kb, 4 Kb, 3 Kb, 2.5 Kb, 2 Kb, 1.5 Kb, 1 Kb) from the top, lanes 2 are primers D1 (SEQ ID NO: 13) and D2 ( About 5.5 Kb of DNA fragments amplified by SEQ ID NO: 14), lane 3, about 5 Kb of DNA fragments amplified by primers E1 (SEQ ID NO: 15) and E2 (SEQ ID NO: 16) were observed.

Figure 3 shows the agarose gel electrophoresis pattern of the gene polymorphism (RFLP) detected by the relative positions of introns A, B, and C on the human airway trypsin enzyme genome, TaqI of intron A and MboI and BstUI of intron C. Indicates.

Herein, the present inventors have made intensive studies in view of such problems of the prior art, and have designed a primer for gene amplification of the intron portion on the genome of the human airway trypsin enzyme that is specifically expressed in human airways. The DNA sequences at both ends of the amplified gene fragment and at the exon / intron boundary portion were newly determined and found to have a gene polymorphism in the amplified gene fragment and the newly determined DNA sequence. In addition, the gene polymorphism analysis led to the first finding of a relationship between the genotype of an individual human airway trypsin form enzyme and a disease.

In other words, the present invention provides a method for predicting the constitution, or the effect of treatment on a patient, or the prognosis of treatment for which a specific disease of an individual is prone to occur by genetic polymorphism analysis of human airway trypsin form enzyme. A method for diagnosing abnormalities in the mucociliary defense system by genetic polymorphism analysis of human airway trypsin form enzymes, the constitution of which a particular disease of each individual is likely to develop, or the effect of treatment on a patient, or the prognosis of treatment How to predict.

In addition, the present invention is a gene fragment containing part or all of the nucleotide sequence of the intron of the human airway trypsin form enzyme.

According to the present invention, there is provided a method for predicting the association between an individual's constitution and a specific disease, and a gene fragment or DNA sequence used for genetic analysis thereof.

Specific diseases for which the constitution easy to be manifested by the method of the present invention, or specific diseases for which the effect of the treatment is judged and the prognosis of the treatment are predicted include diseases of the respiratory system, lung cancer, and particularly chronic obstructive pulmonary disease (COPD). Emphysema (PE), sinus bronchiol syndrome, obese pancreatitis bronchitis (DPB), bronchiectasis (BE), or mucosal ciliary defense system abnormalities are exemplified.

Genetic polymorphism analysis method of the human airway trypsin type enzyme of the present invention, the analysis of genotypes classified by detecting one or more base mutations, and the analysis of the haplotypes classified by detecting one or more base variants Is illustrated.

Genetic polymorphism analysis of the airway trypsin-type enzyme is carried out by an analysis method by Restriction Fragment Length Polymorphism (RFLP), for example, by restriction enzyme digestion. The gene polymorphism analysis method in the present invention also includes a gene polymorphism analysis method which can be detected by Southern hybridization using the cDNA sequence of the human airway trypsin type enzyme. In other words, genomic DNA was digested with a restriction enzyme capable of detecting the gene polymorphism disclosed in the present invention, followed by gel electrophoresis, transcription onto nitrocellulose membrane, and the like, followed by human airway trypsin-type enzyme cDNA as a probe. The cleaved pattern of the human airway trypsin-like enzyme genome is interpreted by Southern hybridization. In addition, the DNA fragments are amplified by PCR so as to include the polymorphic region of the gene, and then analyzed by the method of analyzing the difference by the mobility of electrophoresis (PCR-SSCP (Single Strand Conformation Polymorphism) method), etc. This is possible. As a method of detecting a polymorphic site, mismatch PCR method, PCR-ASO (Allele Specific Oligo) method using allyl specific oligonucleotide, method of annealing using oligoprobe, and determination of direct polymorphism Many methods, such as the pinpoint sequencing method which determines the base sequence of a site | part, can be applied also to the gene diagnosis using a DNA chip.

An intron may be sufficient as a site of gene polymorphism analysis, and the following gene fragment is illustrated as a site of specific gene polymorphism analysis.

(a) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8

(b) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10

(c) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12

(d) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14

(e) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 15 and SEQ ID NO: 16

(f) a gene fragment comprising intron C that can be amplified using the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12

(g) Gene fragments comprising intron A that can be amplified using the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8

Here, as a site for gene polymorphism analysis, a gene fragment containing a sequence portion recognized by restriction enzyme BstUI, MboI, MseI, or FbaI in intron C, and restriction enzyme MboI, TaqI or AfaI in intron A is recognized. One or more combinations of the portion comprising the sequence portion to be made, the polymorphic region of genes shown in SEQ ID NO: 17 are exemplified. By analyzing these sites, genotype or haplotype classification is determined.

In addition, the present invention is a gene fragment comprising part or all of the nucleotide sequence of the intron of the human airway trypsin form enzyme, but in particular the following gene fragments are exemplified.

(a) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8

(b) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10

(c) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12

(d) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14

(e) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 15 and SEQ ID NO: 16

(f) a gene fragment of an intron of a human airway trypsin enzyme consisting of the nucleotide sequences shown in any one of SEQ ID NOs: 1 or 6, or a gene fragment comprising an intron region sandwiched by their nucleotide sequences

(g) Gene fragment of human airway trypsin form enzyme intron C consisting of the nucleotide sequence shown in SEQ ID NO: 17

Example

Hereinafter, although it demonstrates in detail by an Example, the disease related constitution prediction method by the gene polymorphism analysis of this human airway trypsin type enzyme is not limited to this.

Standard operation

Hereinafter, the standard DNA extraction operation, gene amplification operation, restriction enzyme cleavage operation and electrophoresis operation that can be used in the present invention will be described.

(a) standard DNA  Extraction operation

Although it does not specifically limit to the biological sample used for the gene amplification of this invention, It is suitable because a blood cell component is easy to collect a sample and DNA is easy to extract.

1.Put 0.5 ml of whole blood (using 2Na-EDTA anticoagulant) into a 1.5 ml micro centrifuge tube.

2. Add 0.5 ml of solution and invert the tube several times to mix the solution.

(The following mixing operation follows)

Melting example: 1 × SSC

It contains 175.3 g of NaCl and 88.2 g of sodium citrate in 1 liter.

10 × 20 × SSC adjusted to pH 7.0 with 10N NaOH

It is diluted.

3. After centrifugation (10,000 g, 20 sec, 4 ° C.), remove the supernatant so that black pellets do not spill.

4. Add 1 ml of the solution and stir.

5. After centrifugation (10,000 g, 20 sec, 4 ° C.), remove the supernatant.

6. Repeat steps 4 to 5 once more.

7. Add 200 µl of enzyme reaction solution and 10 µl of protease and mix.

Enzyme reaction example: 0.04 M DTT (dithiothritol), 0.2 M NaOAc

Example of a mixed solution protease of (sodium acetate) and 0.4% SDS:

10 mg / ml proteinase (Proteinase K)

8. Keep warm at 37 ° C for 1 hour. (Shake two or three times during mixing.)

9. Add 300 µl of sodium iodide solution and mix.

10. Add 0.5 ml of isopropyl alcohol and mix until white linear DNA is completely visible.

11. Centrifuge (10,000 g, 10 min, room temperature) and remove the supernatant slowly. Remove the remaining solution from the wall of the container by placing the container upside down on the filter paper.

12. 1 ml of washing liquid (A) is added and mixed. Mix enough to allow precipitation to separate from the vessel wall.

Cleaning solution (A) Example: 70% EtOH

13. After centrifugation (10,000 g, 5 min, room temperature), remove the supernatant.

14. 1 ml of washing liquid (B) is added and mixed. Mix enough to allow precipitation to separate from the vessel wall.

Cleaning solution (B) Example: 80% EtOH

15. After centrifugation (10,000 g, 5 min, room temperature), remove the supernatant.

16. Lightly dry the DNA precipitate under reduced pressure. (If it is too dry, DNA will not be dissolved so drying time should be within 3 minutes.)

(b) standard gene amplification

Several principles are known for the gene amplification method, but the polymerase chain reaction method (PCR method) is described here as a standard.

Reaction composition: 50 mM KCl, 10 mM Tris-HCl (pH 9.0, 25 ° C.), 0.1% TritonX-100, 1.5 mM MgCl ₂ , 2 mM dNTPs, 15 μM Forward Primer, 15 μM Reverse Primer ( Riverse Primer), 1 mg / l genomic DNA, 1 unit TaqDNA polymerase, 50 μl total volume

Reaction cycle: 94 ° C., 1 min; 64 ° C., 1 minute; 40 cycles are carried out at 72 ° C for 1 minute at 1 cycle.

As the primer to be used, the following C1 (35 bases) and C2 (35 bases) are used.

C1: 5'-GGAGC CATCT TGTCT GGAAT GCTGT GTGCT GGAGT-3 '

C2: 5'-CACAA TAAAC CAAAG CCGCC GTGAG TCTTC TTGTA-3 '

(c) standard restriction enzyme cleavage

Reaction composition for MboI: 10 mM Tris-HCl (pH 7.4), 10 mM MgCl ₂ , 100 mM NaCl, 10 mM KCl, 1 mM DTT, 100 μg / ml BSA (bovine serum albumin)

MboI is added at a concentration of 10 units / 20 μl reaction solution and incubated at 37 ° C. for 3 hours.

(d) standard electrophoretic manipulation

The electrophoretic buffer is 0.5 × TBE. However, 5 * TBE contains 54 g of Tris bases, 27.5 g of boric acid, and 1 mM EDTA in 1 L, and adjusts pH to 8.0. Using this buffer, it is run for 30 minutes at a voltage of 100 V using 1% or 3% agarose gel (using Seakem GTA Agarose (FMC Bio Products)) (containing 0.5 μg / ml of ethidium bromide). The band of DNA is then observed with a UV lamp.

[ Example  1] on the Human Airway Trypsin Enzyme Genome Intron  Containing DNA  Acquisition by Gene Amplification of Fragments and Analysis of Their Sequences

In order to search for intron sites on the genome of mature human airway trypsin-type enzymes, primers were prepared by referring to the composition of exons and introns of several genomes of tryptase-like enzymes, and amplified human genomic DNA. DNA fragment (gene fragment) of about 6 Kb by A1 (SEQ ID NO: 7) and A2 (SEQ ID NO: 8), about 1.5 Kb by primers B1 (SEQ ID NO: 9) and B2 (SEQ ID NO: 10) DNA fragments of about 3.4 Kb were amplified by primers C1 (SEQ ID NO: 11) and C2 (SEQ ID NO: 12) (Fig. 1).

These DNA fragments are cut out from the gel and purified, and the respective fragments are inserted into a TA cloning vector (Invitrogen). The nucleotide sequence of both sides (5 'end, 3' end) of the insert DNA was determined for some of the clones, followed by the sequence of the primer followed by the cDNA sequence of the human airway trypsin enzyme, followed by the 5 'end, Both sequences of the 3 'end showed the common sequence in the boundary region of exon-intron, and it turned out that the amplified DNA fragment was a DNA fragment including the intron region of human airway trypsin type enzyme.

The sequence of the intron of the gene region encoding the mature protein of the human airway trypsin form enzyme clarified in the present invention will be referred to as intron A, intron B and intron C, respectively, from the 5 'side (Fig. 3). The nucleotide sequences at the 5 'end and the 3' end of each intron which are clarified are as shown by SEQ ID NO: 1, 2, 3, 4, 5, 6. Regarding intron C, the entire nucleotide sequence is shown in SEQ ID NO: 17.

On the other hand, with respect to the region coding for the propeptide of the human airway trypsin enzyme, some primers were prepared and gene amplification was performed. As a result, the primers D1 (SEQ ID NO: 13) and D2 (SEQ ID NO: 14) were about 5.5 Kb. It was clear that about 5 Kb of DNA fragments were amplified by the DNA fragments, primers E1 (SEQ ID NO: 15) and E2 (SEQ ID NO: 16). These gene fragments are thought to contain introns.

[ Example  2] of human airway trypsin form enzyme On intron  Analysis of gene polymorphism

To examine the presence of the gene polymorphisms in introns A, B, and C, 23 healthy genomic DNAs were extracted from whole blood (standard manipulation), and these were respectively amplified primers A1, A2 and intron B for intron A amplification. PCR amplification was carried out using primers B1, B2 and intron C amplification primers C1 and C2, and the cleavage patterns by various restriction enzymes were compared. As a result, in intron A, gene polymorphisms were observed by restriction enzymes MboI, TaqI and AfaI.

In contrast, no gene polymorphism was observed by restriction enzymes Tsp509I, AluI, NlaIII, MspI, BstUI, BfaI, HinPI, HaeIII, HindIII, SspI, PstI, EcoRI, SalI, EcoRV.

For intron B, restriction enzymes Tsp509I, AluI, NlaIII, MspI, BstUI, BfaI, HinPI, HaeIII, MboI, AfaI, TaqI, MseI, ClaI, NsiI, EcoT14I, NdeI, PmlI, ApaLI, AatII, ApaI, KsmI, BsmI Genetic polymorphism was not observed by, HindIII, SspI, EcoRV.

In intron C, gene polymorphisms were observed by restriction enzymes MboI, BstUI, MseI, and FbaI. In contrast, no gene polymorphism was observed by restriction enzymes AluI, NlaIII, MspI, BfaI, HinPI, HaeIII, AfaI, TaqI, HindIII, SspI, BglII, EcoT14I, PvuII, PvuI, EcoRI, BamHI, EcoRV, KpnI.

When DNA fragments containing intron C amplified using a combination of C1-C2 primers were cleaved by MboI or BstUI, in the experiment for cleavage by MboI, bands appeared at 1.3 Kb by electrophoresis. In the case of genotype MM, 1.05 Kb, a band appears in genotype mm, 1.3 Kb and 1.05 Kb, and genotype Mm can be judged. In the case of BstUI, BB is 3.4 Kb and bb 2.45 Kb and 0.95 Kb, all three of which are Bb.

The electrophoretic pattern of the gene polymorphism detected by TaqI of Intron A and MboI and BstUI of Intron C is shown in FIG. 3. In addition, for intron C, the entire nucleotide sequence of the bm-type halotype was determined from DNA fragments obtained by PCR in the genome of a healthy person having an example bbmm type (SEQ ID NO: 17). In the nucleotide sequence of intron C, the polymorphic region detected by BstUI and MboI is shown by the white arrow.

As a result of sequencing of the genome of a patient with a BBmm type BE, the nucleotide sequence of the Bst half-type BstUI gene polymorphic region was found not to be cleaved by BstUI because CGCG became ACCG.

[ Example  3] of human airway trypsin form enzyme Intron  Analysis of Disease Related Constitutions by Genetic Polymorphism Analysis

Regarding the statistical analysis, the analysis was performed by chi-square test using statistical analysis software Stat View 4.02 (Abacus Concepts).

Example  3-1 Interpretation of Disease Related Constitutions by Genotyping

Of the polymorphisms disclosed in Example 2, it was examined whether or not the diseases associated with human airway trypsin enzymes could be classified with respect to the gene polymorphisms detected by MboI and BstUI in intron C. Diseases selected as subjects are obese pancreatitis of bronchial disease (DPB), bronchiectasis (BE), emphysema (PE), and bronchial asthma (BA).

Choose 106 healthy, 29 obese pancreatitis (DPB), 38 bronchiectasis (BE), 22 emphysema (PE), 32 bronchial asthma (BA) and genotype each person according to standard manipulations Was determined. Restriction enzymes were MboI and BstUI.

The appearance factor and frequency of occurrence of each genotype, the appearance frequency and frequency of each allyl type were as shown in Tables 1 and 2.

Table 1 BstUI  genotype

Number of appearances Number of occurrences (alyl) BB Bb bb Sum B b Sum Normal DPBBEPE 168115 55121512 359125 106293822 87283722 125303922 212587644 BA 2 12 18 32 16 48 64

Frequency of occurrence (%) Frequency of occurrence (%) BB Bb bb B b Normal DPBBEPE 15.127.628.922.7 51.941.439.554.5 33.031.031.622.7 41.048.348.750.0 59.051.751.350.0 BA 6.3 37.5 56.3 25.0 75.0

TABLE 2 MboI  genotype

Number of appearances Number of occurrences (alyl) MM Mm mm Sum M m Sum Normal DPBBEPE 10333 407156 56192013 106293822 60132112 152455532 212587644 BA One 12 19 32 14 50 64

Frequency of occurrence (%) Frequency of occurrence (%) MM Mm mm M m Normal DPBBEPE 9.410.37.913.6 37.724.139.527.3 52.865.552.659.1 28.322.427.627.3 71.777.672.472.7 BA 3.1 37.5 59.4 21.9 78.1

From Table 1 and Table 2, it can be seen from the above-described determination of the genotype that the appearance frequency of the BB type is high in DPB, BE, and PE. That is, it is shown that individuals with this genotype can be judged to be constitutions that are likely to become DPB, BE, and PE. In addition, when DPB, BE, and PE were grouped as a group of patients with respiratory tract disease in the category of chronic obstructive disease (COPD), the results showed that the incidence of BB type was statistically higher than that of healthy individuals (chi 2 win p). Value = 0.04, chi 2 power = 4.2).

Frequency of Observation: 3 Diseases, BB / Not BB

BB Not BB Sum normal 16 90 106 Respiratory Diseases 3 24 65 89 Sum 40 155 195

Percentage (row): 3 Diseases, BB / Not BB

BB Not BB Sum normal 15 85 100 Respiratory Diseases 3 27 73 100 Sum 21 79 100

Contingent Table Analysis Statistics: 3 Diseases, BB / Not BB

Missing values 63 Degrees of freedom One Chi 2 Win 4.182 Chi 2 win p value .0409 G2 Win 4.177 G2 win p value .0410 Split Table Analysis Factor .145 Pie (diameter) .146 Chi 2 Wins (Yates Correction) 3.489 Chi 2 Win p Value (Yates Correction) .0618 Fisher's direct method p value .0503

Example  3-2 In the half-ro type classification  Analysis of disease-related constitution

Of the half low type  Frequency of occurrence

By combination of both BstUI and MboI genotypes in 106 healthy, 29 obese pancreatitis (DPB), 38 bronchiectasis (BE), 22 emphysema (PE), and 32 bronchial asthma (BA) The appearance factor and the frequency of occurrence of the halo type are shown in the table. As a result of the survey of 238, the presence of no genotype of Bb-MM, bb-MM and bb-Mm suggests that there is almost no bM haplotype in Japanese.

  Number of appearances BB-MM BB-Nm BB-mm Bb-MM Bb-Mm Bb-mm bb-MM bb-Mm bb-mm Sum normal 10 6 0 0 34 21 0 0 35 106 DPBBEPE 333 270 312 000 586 776 000 000 9125 293822 BA One One 0 0 11 One 0 0 18 32

  Frequency of occurrence (%) BB-MM BB-Nm BB-mm Bb-MM Bb-Mm Bb-mm bb-MM bb-Mm bb-mm Sum normal 9.4 5.7 0.0 0.0 32.1 19.8 0.0 0.0 33.0 100% DPBBEPE 10.37.913.6 6.918.40.0 10.32.69.1 0.00.00.0 17.221.127.3 24.118.427.3 0.00.00.0 0.00.00.0 31.031.622.7 100% 100% 100% BA 3.1 3.1 0.0 0.0 34.4 3.1 0.0 0.0 56.3 100%

Regarding the following interpretations, assuming that the Japanese human airway trypsin type enzyme gene halotype is of three types, BM, Bm, and bm, the correlation with respiratory disease is analyzed using a statistical method (chi 2). Proceeded. (The p values below represent the chi-square p value when the number of occurrences is 5 or more, and Fisher's direct method p value for a 2x2 table containing frames with the number of appearances of 4 or less.)

Allyl Classifieds (1)

As a result of investigating the frequency of occurrence of allyl in each of the three haplotypes of the Japanese, statistically significant differences were observed in the frequency of occurrence between the respiratory 3 disease group (DPB, BE, PE) belonging to COPD and healthy individuals. There was bias (p = 0.027). In particular, Bm allyl had a higher frequency of appearance in the respiratory 3 disease group (DPB, BE, PE) belonging to COPD than healthy persons.

In terms of disease, the number of Bm allyl in BE, DPB, and PE was high.

Frequency of Observation: 3 Diseases, Allyl

Bm. BM b.m. Sum normal 27 60 125 212 Respiratory Diseases 3 41 46 91 178 Sum 68 106 216 390

Percent (row): 3 disease, allyl

Bm. BM b.m. Sum normal 13 28 59 100 Respiratory Diseases 3 23 26 51 100 Sum 17 27 55 100

Contingency Table Analysis Statistics: 3 Diseases, Allyl

Missing values 107 Degrees of freedom 2 Chi 2 Win 7.174 Chi 2 win p value .0277 G2 Win 7.164 G2 win p value .0278 Split Table Analysis Factor .134 V value of Cramer .136

Observation Frequency: DPB Allyl

Bm. BM b.m. Sum normal 27 60 125 212 DPB 15 13 30 58 Sum 42 73 155 270

Percentage (row): DPB Allyl

Bm. BM b.m. Sum normal 13 28 59 100 DPB 26 22 52 100 Sum 16 27 57 100

Split Table Analysis Statistics: DPB Allyl

Missing values 227 Degrees of freedom 2 Year 2 win value 6.044 Year 2 power p value .0487 G2 Win 5.488 G2 win p value .0643 Split Table Analysis Factor .148 V value of Cramer .150

Observation Frequency: BE Allyl

Bm. BM b.m. Sum BE 16 21 39 76 normal 27 60 125 212 Sum 43 81 164 288

Percentage (row): BE Allyl

Bm. BM b.m. Sum BE 21 28 51 100 normal 13 28 59 100 Sum 15 28 57 100

Split Table Analysis Statistics: BE Allyl

Missing values 209 Degrees of freedom 2 Chi 2 Win 3.175 Chi 2 win p value .2044 G2 Win 3.007 G2 win p value .2224 Split Table Analysis Factor .104 V value of Cramer .105

Observation Frequency: PE Allyl

Bm. BM b.m. Sum PE 10 12 22 44 normal 27 60 125 212 Sum 37 72 147 256

Percentage (row): PE Allyl

Bm. BM b.m. Sum PE 23 27 50 100 normal 13 28 59 100 Sum 14 28 57 100

Contingency Table Analysis Statistics: PE Allyl

Missing values 241 Degrees of freedom 2 Chi 2 Win 3.040 Chi 2 win p value .2187 G2 Win 2.765 G2 win p value .2510 Split Table Analysis Factor .108 V value of Cramer .109

Observation Frequency: BA Allyl

Bm. BM b.m. Sum BA 2 14 48 64 normal 27 60 125 212 Sum 29 74 173 276

Percentage (row): BA Allyl

Bm. BM b.m. Sum BA 3 22 75 100 normal 13 28 59 100 Sum 11 27 63 100

Split Table Analysis Statistics: BA Allyl

Missing values 221 Degrees of freedom 2 Chi 2 Win 7.096 Chi 2 win p value .0288 G2 Win 8.264 G2 win p value .0160 Split Table Analysis Factor .158 V value of Cramer .160

Allyl Classifieds (2)

Since Bm allyl is considered to be strongly related to the disease, the relationship between the disease and the number of allyls of the Bm type and the number of allyls other than Bm has been analyzed.

In the respiratory 3 disease group (DPB, BE, PE) belonging to COPD, when the healthy group and the respiratory 3 disease group are compared, the occurrence frequency of Bm allyl in the respiratory 3 disease group is certainly high, and the bias of distribution There was a statistically significant difference (p = 0.002). This comparison showed a relationship between the Bm-type allyl and the onset of respiratory 3 disease group (DPE, BE, PE) belonging to COPD.

In terms of disease, the respiratory 3 disease group (DPB, BE, PE) belonging to COPD had higher frequency of Bm type than normal patients. (BE; p = 0.012, DPB; p = 0.0025, PE; p = 0.0052)

On the other hand, in BA, the frequency of appearance of Bm type was significantly lower than that of healthy persons. (p = 0.049)

Frequency of Observation: 3 Diseases, Bm / Not Bm

Bm Not Bm Sum normal 27 185 212 Respiratory Diseases 3 49 129 178 Sum 76 314 390

Percentage (row): 3 Diseases, Bm / Not Bm

Bm Not Bm Sum normal 13 87 100 Respiratory Diseases 3 28 72 100 Sum 19 81 100

Contingent Table Analysis Statistics: 3 Diseases, Bm / Not Bm

Missing values 107 Degrees of freedom One Chi 2 Win 13.494 Chi 2 win p value .0002 G2 Win 13.534 G2 win p value .0002 Split Table Analysis Factor .183 pie .186 Chi 2 Wins (Yates Correction) 12.568 Chi 2 Win p Value (Yates Correction) .0004 Fisher's direct method p value .0003

Observation frequency: DPB, Bm / Not Bm

Bm Not Bm Sum normal 27 185 212 DPB 17 41 58 Sum 44 226 270

Percentage (row): DPB, Bm / Not Bm

Bm Not Bm Sum normal 13 87 100 DPB 29 71 100 Sum 16 84 100

Split Table Analysis Statistics: DPB, Bm / Not Bm

Missing values 227 Degrees of freedom One Chi 2 Win 9.172 Chi 2 win p value .0025 G2 Win 8.202 G2 win p value .0042 Split Table Analysis Factor .181 pie .184 Chi 2 Wins (Yates Correction) 7.997 Chi 2 Win p Value (Yates Correction) .0047 Fisher's direct method p value .0045

Observation frequency: BE, Bm / Not Bm

Bm Not Bm Sum BE 19 57 76 normal 27 185 212 Sum 46 242 288

Percentage (row): BE, Bm / Not Bm

Bm Not Bm Sum BE 25 75 100 normal 13 87 100 Sum 16 84 100

Split Table Analysis Statistics: BE, Bm / Not Bm

Missing values 209 Degrees of freedom One Chi 2 Win 6.270 Chi 2 win p value .0123 G2 Win 5.824 G2 win p value .0158 Split Table Analysis Factor .146 pie .148 Chi 2 Wins (Yates Correction) 5.389 Chi 2 Win p Value (Yates Correction) .0203 Fisher's direct method p value .0172

Observation frequency: PE, Bm / Not Bm

Bm Not Bm Sum PE 13 31 44 normal 27 185 212 Sum 40 216 256

Percentage (row): PE, Bm / Not Bm

Bm Not Bm Sum PE 30 70 100 normal 13 87 100 Sum 16 84 100

Split Table Analysis Statistics: PE, Bm / Not Bm

Missing values 241 Degrees of freedom One Chi 2 Win 7.810 Chi 2 win p value .0052 G2 Win 6.802 G2 win p value .0091 Split Table Analysis Factor .172 pie .175 Chi 2 Wins (Yates Correction) 6.587 Chi 2 Win p Value (Yates Correction) .0103 Fisher's direct method p value .0104

Observation frequency: BA, Bm / Not Bm

Bm Not Bm Sum BA 2 62 64 normal 27 185 212 Sum 29 247 276

Percentage (row): BA, Bm / Not Bm

Bm Not Bm Sum BA 3 97 100 normal 13 87 100 Sum 11 89 100

Split Table Analysis Statistics: BA, Bm / Not Bm

Missing values 221 Degrees of freedom One Chi 2 Win 4.829 Chi 2 win p value .0280 G2 Win 6.035 G2 win p value .0140 Split Table Analysis Factor .131 pie .132 Chi 2 Wins (Yates Correction) 3.861 Chi 2 Win p Value (Yates Correction) .0494 Fisher's direct method p value .0340

Classification (1)

From the analysis results of the allyl classification, it is thought that the Bm type among the three halotypes is particularly related to the respiratory disease. The individual (homo) having it was classified and analyzed.

In the respiratory 3 disease group belonging to COPD (DPB, BE, PE), when comparing the healthy group with the respiratory 3 disease group, regarding the frequency of the halotype classification Bm-0, 1, 2 based on Bm, There was a significant difference in the distribution (p = 0.0093).

By disease, there was a significant difference in the distribution of DPB and PE patients (DPB; p = 0.0024, PE; p = 0.0069) and in BE (p = 0.089).

Frequency of Observation: 3 Disease, Bm.

Bm-0 Bm-1 Bm-2 Sum normal 79 27 0 106 Respiratory Diseases 3 54 29 6 89 Sum 133 56 6 195

Percent (row): 3 disease, Bm.

Bm-0 Bm-1 Bm-2 Sum normal 75 25 0 100 Respiratory Diseases 3 61 33 7 100 Sum 68 29 3 100

Contingent Table Analysis Statistics: 3 Diseases, Bm.

Missing values 63 Degrees of freedom 2 Chi 2 Win 9.360 Chi 2 win p value .0093 G2 Win . G2 win p value . Split Table Analysis Factor .214 V value of Cramer .219

Observation frequency: DPB, Bm.

Bm-0 Bm-1 Bm-2 Sum normal 79 27 0 106 DPB 17 9 3 29 Sum 96 36 3 135

Percentage (row): DPB, Bm.

Bm-0 Bm-1 Bm-2 Sum normal 75 25 0 100 DPB 59 31 10 100 Sum 71 27 2 100

Split Table Analysis Statistics: DPB, Bm.

Missing values 123 Degrees of freedom 2 Chi 2 Win 12.040 Chi 2 win p value .0024 G2 Win . G2 win p value . Split Table Analysis Factor .286 V value of Cramer .299

Observation frequency: BE, Bm.

Bm-0 Bm-1 Bm-2 Sum BE 23 14 One 38 normal 79 27 0 106 Sum 102 41 One 144

Percentage (row): BE, Bm.

Bm-0 Bm-1 Bm-2 Sum BE 61 37 3 100 normal 75 25 0 100 Sum 71 28 One 100

Split Table Analysis Statistics: BE, Bm.

Missing values 114 Degrees of freedom 2 Chi 2 Win 4.834 Chi 2 win p value .0892 G2 Win . G2 win p value . Split Table Analysis Factor .180 V value of Cramer .183

Observation frequency: PE, Bm.

Bm-0 Bm-1 Bm-2 Sum PE 14 6 2 22 normal 79 27 0 106 Sum 93 33 2 128

Percent (row): PE, Bm.

Bm-0 Bm-1 Bm-2 Sum PE 64 27 9 100 normal 75 25 0 100 Sum 73 26 2 100

Contingent Table Analysis Statistics: PE, Bm.

Missing values 130 Degrees of freedom 2 Chi 2 Win 9.957 Chi 2 win p value .0069 G2 Win . G2 win p value . Split Table Analysis Factor .269 V value of Cramer .279

Classification (2)

It was analyzed whether it had Bm haplotype (Bm-0 vs Bm-1, 2).

In the respiratory 3 disease group belonging to COPD (DPB, BE, PE), compared with healthy people, the occurrence of Bm in the respiratory 3 disease group was high and there was a significant difference in the distribution bias. (P = 0.039). On the other hand, in BA, many individuals did not have the Bm halflotype, and showed a significant bias in distribution compared with the healthy group (p = 0.037).

Frequency of Observation: 3 Diseases, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum normal 79 27 106 Respiratory Diseases 3 54 35 89 Sum 133 62 195

Percentage (row): 3 Diseases, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum normal 75 25 100 Respiratory Diseases 3 61 39 100 Sum 68 32 100

Contingent Table Analysis Statistics: Three Diseases, Bm-0 / Bm1.2

Missing values 63 Degrees of freedom One Chi 2 Win 4.282 Chi 2 win p value .0385 G2 Win 4.279 G2 win p value .0386 Split Table Analysis Factor .147 pie .148 Chi 2 Wins (Yates Correction) 3.670 Chi 2 Win p Value (Yates Correction) .0554 Fisher's direct method p value .0453

Observation frequency: BA, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum BA 30 2 32 normal 79 27 106 Sum 109 29 138

Percentage (row): BA, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum BA 94 6 100 normal 75 25 100 Sum 79 21 100

Split Table Analysis Statistics: BA, Bm-0 / Bm1.2

Missing values 120 Degrees of freedom One Chi 2 Win 5.471 Chi 2 win p value .0193 G2 Win 6.641 G2 win p value .0100 Split Table Analysis Factor .195 pie .199 Chi 2 Wins (Yates Correction) 4.375 Chi 2 Win p Value (Yates Correction) .0365 Fisher's direct method p value .0241

Observation frequency: DPB, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum normal 79 27 106 DPB 17 12 29 Sum 96 39 135

Percentage (row): DPB, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum normal 75 25 100 DPB 59 41 100 Sum 71 29 100

Split Table Analysis Statistics: DPB, Bm-0 / Bm1.2

Missing values 123 Degrees of freedom One Chi 2 Win 2.805 Chi 2 win p value .0940 G2 Win 2.674 G2 win p value .1020 Split Table Analysis Factor .143 pie .144 Chi 2 Wins (Yates Correction) 2.089 Chi 2 Win p Value (Yates Correction) .1484 Fisher's direct method p value .1086

Observation frequency: BE, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum BE 23 15 38 normal 79 27 106 Sum 102 42 144

Percentage (row): BE, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum BE 61 39 100 normal 75 25 100 Sum 71 29 100

Split Table Analysis Statistics: BE, Bm-0 / Bm1.2

Missing values 114 Degrees of freedom One Chi 2 Win 2.654 Chi 2 win p value .1033 G2 Win 2.564 G2 win p value .1093 Split Table Analysis Factor .135 pie .136 Chi 2 Wins (Yates Correction) 2.020 Chi 2 Win p Value (Yates Correction) .1552 Fisher's direct method p value .1444

Observation frequency: PE, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum PE 14 8 22 normal 79 27 106 Sum 93 35 128

Percentage (row): PE, Bm-0 / Bm1.2

Bm-0 Bm-1.2 Sum PE 64 36 100 normal 75 25 100 Sum 73 27 100

Split Table Analysis Statistics: PE, Bm-0 / Bm1.2

Missing values 130 Degrees of freedom One Chi 2 Win 1.088 Chi 2 win p value .2969 G2 Win 1.040 G2 win p value .3079 Split Table Analysis Factor .092 pie .092 Chi 2 Wins (Yates Correction) .609 Chi 2 Win p Value (Yates Correction) .4353 Fisher's direct method p value .3035

Classification (3)

In addition, the frequency of occurrence was compared between individuals with Bm haplotype (BBmm; Bm-2) and those without (Bm-0, 1) (Bm-0, 1 vs Bm-2).

In the respiratory 3 disease group belonging to COPD (DPB, BE, PE), compared to healthy individuals, individuals with Bm haplotype homozygous (BBmm) and those without it had a bias in distribution with respect to frequency of appearance. Statistically significant difference (p = 0.021), all 6 of the Bm homotypes (Bm-2) are remorseful to any one of the respiratory 3 diseases (DPB, BE, PE) belonging to COPD, and three of them are DPB 2 were PE and 1 was BE. There were no Bm homotypes (Bm-2) among the 106 healthy and 32 BA.

By disease, the patients with DPB and PE showed statistically significant differences in the distribution (DPB; p = 0.0082, PE; p = 0.0029).

Frequency of Observation: 3 Diseases, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum normal 106 0 106 Respiratory Diseases 3 83 6 89 Sum 189 6 195

Percentage (row): 3 Diseases, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum normal 100 0 100 Respiratory Diseases 3 93 7 100 Sum 97 3 100

Contingent Table Analysis Statistics: Three Diseases, Bm-0.1 / Bm-2

Missing values 63 Degrees of freedom One Chi 2 Win 7.373 Chi 2 win p value .0066 G2 Win . G2 win p value . Split Table Analysis Factor .191 pie .194 Chi 2 Wins (Yates Correction) 5.295 Chi 2 Win p Value (Yates Correction) .0214 Fisher's direct method p value .0082

Observation frequency: DPB, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum normal 106 0 106 DPB 26 3 29 Sum 132 3 135

Percentage (row): DPB, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum normal 100 0 100 DPB 90 10 100 Sum 98 2 100

Split Table Analysis Statistics: DPB, Bm-0.1 / Bm-2

Missing values 123 Degrees of freedom One Chi 2 Win 11.215 Chi 2 win p value .0008 G2 Win . G2 win p value . Split Table Analysis Factor .277 pie .288 Chi 2 Wins (Yates Correction) 6.987 Chi 2 Win p Value (Yates Correction) .0082 Fisher's direct method p value .0091

Observation frequency: PE, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum PE 20 2 22 normal 106 0 106 Sum 126 2 128

Percentage (row): PE, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum PE 91 9 100 normal 100 0 100 Sum 98 2 100

Split Table Analysis Statistics: PE, Bm-0.1 / Bm-2

Missing values 130 Degrees of freedom One Chi 2 Win 9.789 Chi 2 win p value .0018 G2 Win . G2 win p value . Split Table Analysis Factor .267 pie .277 Chi 2 Wins (Yates Correction) 4.771 Chi 2 Win p Value (Yates Correction) .0289 Fisher's direct method p value .0284

Observation frequency: BE, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum BE 37 One 38 normal 106 0 106 Sum 143 One 144

Percentage (row): BE, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum BE 97 3 100 normal 100 0 100 Sum 99 One 100

Split Table Analysis Statistics: BE, Bm-0.1 / Bm-2

Missing values 114 Degrees of freedom One Chi 2 Win 2.809 Chi 2 win p value .0937 G2 Win . G2 win p value . Split Table Analysis Factor .138 pie .140 Chi 2 Wins (Yates Correction) .289 Chi 2 Win p Value (Yates Correction) .5909 Fisher's direct method p value .2639

Observation frequency: BA, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum BA 32 0 32 normal 106 0 106 Sum 138 0 138

Percentage (row): BA, Bm-0.1 / Bm-2

Bm-0.1 Bm-2 Sum BA 100 0 100 normal 100 0 100 Sum 100 0 100

As mentioned above, intron gene polymorphism analysis of human airway trypsin type enzyme revealed that Bm-type halotype is related to chronic airway inflammation in respiratory diseases by any mechanism. In addition, the involvement of the disease of human airway trypsin form enzyme was also different in the three diseases of DPB, BE, PE belonging to chronic obstructive disease (COPD) and BA.

However, since not all individuals with a certain polymorphism develop a disease, the gene polymorphism of the human airway trypsin form enzyme is not a definitive onset factor, such as a hereditary disease, but can be said to be a gradual factor. That is, when environmental factors are added to the sieve having the Bm halflotype, respiratory diseases such as BE, PE, and DPB are more likely to occur.

From these results, it was shown that by using the gene polymorphism of the human airway trypsin form enzyme, it is possible to classify diseases related to human airway trypsin form enzymes.

In addition, the gene polymorphism analysis method of the human airway trypsin type enzyme is used to predict the predisposition of human airway trypsin enzyme related diseases, the effect on the treatment of the disease, the prediction of the possibility of recurrence, etc. It is a means that can be applied.

The present invention provides a method for measuring disease-related constitution of an individual by a method for analyzing polymorphism of human airway trypsin type enzyme. Therefore, if it is possible to identify a disease associated with human airway trypsin enzyme by this interpretation, it can be estimated that an individual having a genotype with the human airway trypsin enzyme is susceptible to certain diseases.

In other words, by predicting the disease constitution (that the individual has a constitution easy to be affected by a certain disease), it becomes possible to give information about the individual's treatment early, leading to early diagnosis and early treatment. In addition, the possibility of the disease recurring even after treatment can be estimated. In other words, by predicting the prognosis of the treatment (the progress of healing after the patient's treatment and the risk of recurrence), the doctor can pay sufficient attention to the patient and provide appropriate guidance.

Furthermore, in a condition involving the human airway trypsin-type enzyme, the gene polymorphism analysis of the human airway trypsin-type enzyme may be a means of estimating the effective patient group of the administered drug in the determination of the effect of the drug to be administered and the drug development. .

As mentioned above, it leads to the reduction of the expensive medical cost currently a problem by early diagnosis, early treatment, instruction of appropriate preventive method, appropriate administration, and later management after treatment.

Sequence table

SEQ ID NO: 1

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron A 5 'end

order:

SEQ ID NO: 2

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron A 3 'end

order:

SEQ ID NO: 3

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron B 5 'end

order:

SEQ ID NO: 4

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron B 3 'end

order:

SEQ ID NO: 5

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron C 5 'end

order:

SEQ ID NO: 6

Length of sequence:

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: intron C 3 'end

order:

SEQ ID NO: 7

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: A1

order:

SEQ ID NO: 8

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: A2

order:

SEQ ID NO: 9

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: B1

order:

SEQ ID NO: 10

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: B2

order:

SEQ ID NO: 11

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: C1

order:

SEQ ID NO: 12

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: C2

order:

SEQ ID NO: 13

Length of sequence:

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: D1

order:

SEQ ID NO: 14

Length of sequence:

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: D2

order:

SEQ ID NO: 15

Length of sequence:

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: E1

order:

SEQ ID NO: 16

Length of sequence:

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of sequence: nucleic acid (primer)

Characteristic of sequence: E2

order:

SEQ ID NO: 17

Length of sequence: 3234

Type of sequence: nucleic acid

Number of chains: two chain

Topology: linear

Type of sequence: nucleic acid (genomic sequence)

Characteristic of sequence: Intron C

order:

Claims (27)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete One of the following gene fragments: (a) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8, (b) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10, (c) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12, (d) a gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14, (e) A gene fragment comprising an intron region that can be amplified using the primers shown in SEQ ID NO: 15 and SEQ ID NO: 16. delete A gene fragment of an intron of a human airway trypsin form enzyme consisting of the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6. A gene fragment of the human airway trypsin form enzyme intron C consisting of the nucleotide sequence shown in SEQ ID NO: 17. A primer pair consisting of the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 which can be used for amplification of the gene for detecting MboI restriction enzyme fragment fragment length (RFLP) or BstUI RFLP in intron C of the human airway trypsin form enzyme gene. ).